Anti epas 1 hif 2 alpha

Cells were transfected with appropriate plasmids and lysed by co-immunoprecipitation lysis buffer (10% glycerol, 0.5% NP-40, 150 mM NaCl, 0.1 mM EDTA) supplemented with protease inhibitor cocktail (Roche). Cell lysates were incubated with the S-protein Agarose beads (Millipore, 69704) or indicated primary antibody and protein A/G agarose beads (Santa Cruz Biotechnology, sc-2003). The immunocomplexes were then washed by PBSN (PBS containing 0.1% NP-40) three times and subjected to SDS-Page. For subcellular fractionation, nuclear and cytoplasmic extracts were isolated with a nuclear-cytoplasmic extraction kit (Applygen, P1200) following the manufacturer’s protocol.
Antibodies used in this study were as follows: anti-VHL (Abcam, ab77262) for Western Blot, anti-VHL (Abclonal, A0377) for Immunohistochemistry, anti- EPAS-1/HIF-2 alpha (Santa Cruz Biotechnology, sc-13596), anti-BICD2 (Abcam, ab237616), anti-GAPDH (TransGen Biotech, HC301-01), anti-β-Tubulin (ABclonal, AC021), anti-β-Actin (ABclonal, AC004), anti-HDAC1 (ABclonal, A19571), anti-FLAG (Sigma, F3165), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti-GFP (MBL, 598).

Cells were transfected with appropriate plasmids and lysed by co-immunoprecipitation lysis buffer (10% glycerol, 0.5% NP-40, 150 mM NaCl, 0.1 mM EDTA) supplemented with protease inhibitor cocktail (Roche). Cell lysates were incubated with the S-protein Agarose beads (Millipore, 69704) or indicated primary antibody and protein A/G agarose beads (Santa Cruz Biotechnology, sc-2003). The immunocomplexes were then washed by PBSN (PBS containing 0.1% NP-40) three times and subjected to SDS-Page. For subcellular fractionation, nuclear and cytoplasmic extracts were isolated with a nuclear-cytoplasmic extraction kit (Applygen, P1200) following the manufacturer’s protocol.
Antibodies used in this study were as follows: anti-VHL (Abcam, ab77262) for Western Blot, anti-VHL (Abclonal, A0377) for Immunohistochemistry, anti- EPAS-1/HIF-2 alpha (Santa Cruz Biotechnology, sc-13596), anti-BICD2 (Abcam, ab237616), anti-GAPDH (TransGen Biotech, HC301-01), anti-β-Tubulin (ABclonal, AC021), anti-β-Actin (ABclonal, AC004), anti-HDAC1 (ABclonal, A19571), anti-FLAG (Sigma, F3165), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti-GFP (MBL, 598).